Search Results (86)

Molecular sex determination in Syrian hamsters (Mesocricetus auratus) has been limited by the incomplete annotation of Y-linked loci in currently available genome assemblies. Here, we evaluate the Y-linked gene PRSSLY, which encodes a testis-specific serine protease-like protein, as a molecular marker for genetic sexing of Syrian hamster embryonic and placental tissues. Primers flanking a conserved PRSSLY coding region produced a male-specific amplicon showing 100% concordance with results from the established KDM5C/KDM5D PCR assay in E15.5 tail biopsies. SYBR Green–based qPCR enables the accurate detection of PRSSLY, characterized by a unique melt-curve profile, exclusively in male samples, allowing for efficient and sensitive mid-throughput analysis. Application of the PRSSLY assay to 417 placental samples from 39 dams demonstrated its suitability for large-scale sex genotyping, enabling sex assignment in the majority of samples despite the intrinsic complexity of placental tissue containing both maternal and embryonic genetic material. This assay provides a robust and reproducible approach for accurate sex genotyping in developmental and reproductive studies using Syrian hamsters. Full article

Heterocapsa bohaiensis is an emerging harmful dinoflagellate increasingly reported from coastal regions of the Pacific. However, an available molecular assay offering rapid and sensitive detection is still lacking. This study developed a SYBR Green real-time quantitative PCR (qPCR) assay for the identification and quantification of H. bohaiensis. Species-specific primers (F: 5′-CCATCGAACCAGAACTCCGT-3′; R: 5′-AGTGTAGTGCACCGCATGTC-3′) were designed and the assay was optimized and evaluated using laboratory cultures for specificity, sensitivity, and quantitative performance. Primer screening and melt-curve analysis confirmed that the selected primer pair produced a single, specific amplification peak for H. bohaiensis, with no cross-reactivity observed in non-target species (Chlorella pyrenoidosa, Phaeocystis globosa, Skeletonema costatum, Alexandrium tamarense) or mixed algal communities. The standard curve displayed strong linearity (R² = 0.9868) and a high amplification efficiency (102.5%). The limit of detection (LOD) was approximately 2–3 cells per reaction, as determined from 24 replicates of 5-cell equivalents and verified at ~2.7-cell equivalents. This sensitivity was comparable to or exceeded that reported for assays targeting other HABs forming dinoflagellates. Quantitative results derived from the qPCR assay closely matched microscopic cell counts, with a relative error of 10.79%, falling within the acceptable threshold for phytoplankton surveys. In summary, this study established and validates a species-specific qPCR assay for H. bohaiensis under controlled laboratory conditions. The method shows strong potential for incorporation into HAB monitoring programs, early-warning systems, and future ecological investigations of this emerging species. Full article

In this study, we focused on the screening and identification of reference genes for Paracarophenax alternatus Xu and Zhang. The laboratory population was used as the laboratory population, and samples were collected from mites at four different stages, including physogastry, viviparous, 5 d viviparous and phoresy. Then, the expression levels of seven candidate reference genes (α-tubulin, β-tubulin, RPS18, RPL13, GAPDH, EF1A, SDHA) were detected through qRT-PCR. Melting curves showed good gene specificity, and the amplification efficiency ranged from 90% to 102%. ΔCt analysis indicated that GAPDH was the most stable reference gene. The GeNorm software determined that the optimal number of reference genes was two, with GAPDH and RPS18 forming the most stable combination, and NormFinder identified RPS18 as the most stable reference gene. Although the BestKeeper software suggested that EF1A was the most stable, its p-value exceeded 0.05, rendering it unsuitable for use as a reference gene. Finally, through the RefFinder network tool, the most stable reference genes were identified as GAPDH and RPS18. Full article

Background: The cytochrome P450 family 21 subfamily A member 2 gene (CYP21A2) encodes 21-hydroxylase, a key enzyme in adrenal steroid biosynthesis. Despite its physiological importance, the diversity of CYP21A2 transcript variants and their tissue-specific expression in domestic animals, including cattle, remains largely unexplored. This study aimed to characterize CYP21A2 transcription in adrenal glands and ovaries and assess the potential transcriptional activity of its pseudogene, CYP21A1P. Methods: CYP21A2 transcription was investigated in adrenal and ovarian tissues of 12 healthy cows using semi-quantitative PCR and Sanger sequencing. Real-time PCR was performed to confirm expression levels. Melting curve analysis and electrophoresis were used to validate distinct amplicons corresponding to different transcript variants. Extended amplicons were sequenced to identify transcripts corresponding to reference sequences and potential pseudogene products. Results: A single transcript variant (NM_001013596.1) was consistently detected in adrenal glands, whereas ovaries expressed two variants: NM_001013596.1 and XM_024983378.2. Semi-quantitative analysis showed significantly higher CYP21A2 expression in adrenal glands compared to ovaries (p < 0.01). In ovarian samples, the NM_001013596.1 variant was more abundant than the XM_024983378.2 (p < 0.01). Sanger sequencing revealed two products matching CYP21A2 reference transcripts and an additional, longer product containing sequence motifs specific to the pseudogene CYP21A1P, indicating its transcriptional activity. Conclusions: These results provide the first evidence of tissue-specific expression and differential abundance of CYP21A2 transcript variants in cattle and suggest the transcription of the CYP21A1P pseudogene. The findings reveal the complexity of CYP21A2 expression in steroidogenic tissues and suggest potential regulatory roles for transcript and pseudogene variants in bovine physiology. Full article

Background and Objectives: Thrombophilia is a prothrombotic disorder that increases the risk of blood clotting and can pose serious health problems. It is considered a condition of gene–gene or gene–environment interactions. Variation in the prevalence of thrombophilia mutations and their interaction among populations necessitates localized genetic assessments. However, population-based genetic data remains limited for developing effective preventive strategies. Materials and Methods: This cross-sectional observational study was conducted over five years (2020–2024) at a tertiary university hospital in Northern Greece. A total of 2961 individuals aged 18–85 years (mean: 50.5) were registered based on family or medical history of venous thromboembolism (VTE) or clinical symptoms of VTE. The final analysis included 2078 participants comprising 1143 males (55%) and 935 females (45%), who met all the inclusion criteria. Inclusion criteria were absence of acute illness or malignancy, informed consent, and an adequate DNA quantity for genotyping, whereas excluded criteria included incomplete laboratory data, active inflammatory or malignant disease, and cognitive or psychiatric conditions. Peripheral blood samples were collected in 2 mL K₃-EDTA tubes, and genomic DNA was analyzed using real-time polymerase chain reaction (PCR) with melting curve analysis and hybridization probes (LightMix^® in vitro diagnostics, TIB MolBiol, Berlin, Germany). Five thrombophilia-related polymorphisms, Factor V Leiden (F5 G1691A), prothrombin (F2 G20210A), methylenetetrahydrofolate reductase (MTHFR C677T and MTHFR A1298C), and Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G, were examined for allele and genotype frequencies, Hardy–Weinberg equilibrium testing, pairwise linkage disequilibrium (D′ and r²), and power analysis. For subjects tested for Factor V Leiden (n = 1476), the activated protein C resistance (APC) ratio was additionally evaluated using the ACL TOP 750 analyzer. Results: Allele frequencies were 7.3% for FV Leiden and 3.7% for FII. The PAI-1 allele was distributed at 44%, while the MTHFR (C677T and A1298C) alleles were each present at 33%. Significant linkage disequilibrium was identified between MTHFR (C677T and A1298C) and between MTHFR A1298C and PAI-1. No evolutionary pressure or demographic bias was found in the Hardy–Weinberg equilibrium. The APC ratio demonstrated a high sensitivity (99.2%) and specificity (96.6%), indicating that it may serve as a reliable screening method. Conclusions: Our findings highlight informative patterns in the genetic predisposition to thrombophilia, which may help develop rule-based strategies for implementing thromboprophylaxis guidelines and personalized medical interventions. Full article

Carotid artery intima–media thickness (cIMT), a pre-clinical vascular change that accompanies atherosclerosis is considered as a cardiovascular risk factor. Coagulation factor XIII (FXIII) stabilizes the fibrin clot and increases its resistance to fibrinolysis. Regarding FXIII Val34Leu polymorphism, the protective effect of the Leu34 allele in the presence of elevated fibrinogen levels against myocardial infarction was demonstrated. Our aim was to investigate the effect of FXIII polymorphisms on cIMT. Patients with obesity (n = 69), type 2 diabetes mellitus (T2DM) (n = 104), and age- and sex-matched healthy controls (n = 82) were enrolled. FXIII polymorphisms (Val34Leu, His95Arg, Intron-K C>G) were determined by RT-PCR with FRET detection and melting curve analysis. cIMT was determined by B-mode ultrasound. Differences in cIMT between control (median: 0.5965, IQR: 0.5115–0.6580 mm) and T2DM (median: 0.7105, IQR: 0.5948–0.7568 mm), as well as between obese (median: 0.6105, IQR: 0.5455–0.6780 mm) and diabetic groups, were found (p < 0.0001 and p = 0.003, respectively). Genotype and allele frequencies of the studied polymorphisms did not differ between subgroups. In the study group (n = 255) after adjustment for age and sex, the presence of Intron-K G allele showed a significant and independent protective effect against cIMT progression in a separate model (p = 0.005) and after adjusting for other parameters associated with cIMT (p = 0.015). FXIII Intron-K G allele provides a protective effect against subclinical vascular disease in the studied population, and this effect is independent of the presence of obesity, as well as T2DM, Leu34 allele, and fibrinogen levels. Full article

Salix suchowensis is an ideal model organism for investigating nitrogen (N) transport mechanisms due to its low N-input requirements. Accurate quantification of gene expression is essential for elucidating these processes, with quantitative real-time PCR (RT-qPCR) being the preferred method. However, the identification of stable reference genes for normalization in Salix suchowensis under varying N conditions remains unresolved. In this study, thirteen commonly employed candidate reference genes were evaluated across root, stem, and leaf tissues, under four N treatments (NH₄NO₃, NH₄⁺, NO₃⁻, and N deficiency). Five genes (UBQ1, UBQ3, 18S, H2A2, and H2B2) were excluded due to poor amplification efficiency or irregular melting curves. The remaining eight genes were further assessed for expression stability using the geNorm, NormFinder, and BestKeeper algorithms. Integrated ranking via RefFinder identified EF1α, EFβ, and αTUB as the most stable reference genes. GeNorm analysis suggested that two reference genes were sufficient for reliable normalization. Validation using the N-responsive gene SsAMT1 and SsNRT2 confirmed the stability of EF1α, EFβ, and αTUB as suitable reference genes. Based on comprehensive stability assessments and experimental validation, we recommended EF1α + αTUB as optimal reference gene pairs for RT-qPCR normalization under varying N conditions. Furthermore, the consistent expression of EF1α and αTUB across nine willow genotypes highlighted their broader applicability within Salix species. This study provides valuable methodological guidance for advancing molecular research on N transport in woody perennial plants. Full article

Background/Objectives: Phosphoglucomutase-1 (PGM1) is an enzyme that plays important roles in glycolysis, glycogen metabolism, and glycosylation. The PGM1 gene harbors two common nonsynonymous single-nucleotide variants (rs1126728 and rs11208257), which result in four functional PGM1 phenotypes. Correlations between PGM1 polymorphisms and several pathological conditions have been suggested. Methods: To identify the rs1126728 and rs11208257 concurrently, a fluorescence melting curve analysis (FMCA) was developed that utilizes two distinct dual-labeled fluorescence probes. Two distinct Taq polymerases, one with and one without 5′-3′exonuclease activity, were compared. This method was then applied to 95 unrelated Japanese subjects. Results: Both Taq polymerases, with and without 5′-3′exonuclease activity, were found to be sufficiently functional. Furthermore, the results of the FMCA using both Taq polymerases were compared with the direct Sanger sequencing results of PCR products from the 95 Japanese subjects, demonstrating 100% concordance. Conclusions: The duplex probe-based FMCA developed in this study is useful for examining the association between rs1126728 or rs11208257 and a range of pathological conditions using a relatively large number of subjects. Full article

Background/Objectives: Personality traits influence motivation, self-regulation, and adaptation in high-performance sports, and are partially modulated by dopaminergic genetic variability. This study aimed to examine the association between the DRD2 Ex8 rs6276 polymorphism and NEO Five-Factor Inventory (NEO-FFI) personality traits in elite athletes and non-athlete controls. Methods: A total of 323 participants were included: 141 athletes and 182 controls. Genomic DNA was isolated from venous blood, and DRD2 Ex8 rs6276 genotypes (A/A, A/G, G/G) were determined using real-time PCR with melting-curve analysis. Personality traits were assessed using the NEO-FFI, and group differences as well as genotype × group interactions were evaluated using multivariate analyses and non-parametric tests. Results: Athletes scored significantly higher on Conscientiousness than controls. A genotype × group interaction was observed for Extraversion, and the main effect of the genotype was found to be Agreeableness. Athletes with the A/A genotype exhibited the highest Extraversion scores, whereas those with the G/G genotype demonstrated higher Agreeableness than other genotypes. Conclusions: These findings indicate that dopaminergic variation contributes to individual differences in social and motivational traits, which may support athletic engagement and adaptation to high-demand environments. The results should be interpreted with caution due to the moderate sample size, deviation from the Hardy–Weinberg equilibrium in the athlete group, and reliance on a single personality assessment tool. Full article

Palm lethal yellowing phytoplasmas (PLYPs) are a group of phytoplasmas that cause death in infected hosts across the tropics. Historically, detection and identification has relied on standard PCR, nested PCR, and restriction fragment length polymorphism. While these approaches are generally good, they are prone to error and contamination that is significantly lower or absent in modern approaches using quantitative PCR (qPCR) and digital PCR (dPCR). Additionally, these modern approaches are more time-efficient and consume fewer resources, making them more cost-effective in the long term. Recent studies have adapted dPCR and qPCR coupled with high-resolution melt curve analysis (HRMA) for PLYPs in the Caribbean/New World; however, these tools have not been developed for phytoplasmas in Africa and Madagascar. In this study, a duplex dPCR assay was developed with two specific assays, one for ‘Candidatus Phytoplasma palmicola’ and one for ‘Ca. P. cocostanzaniae’ and isolates from Madagascar. Additionally, primers targeting the secA gene were optimized and allowed for the separation of ‘Ca. P. cocostanzaniae’ and Malagasy isolates by approximately one degree Celsius. New primers were developed based on secA for ‘Ca. P. palmicola’ that allowed for the separation of the two subgroups (A and B) by HRMA by a difference of approximately one degree Celsius. These assays provide a valuable resource to explore aspects such as vector discovery and host range. Full article

Honey is a natural bee product with confirmed health-promoting properties, the quality and authenticity of which are of key importance from a consumer’s perspective. However, the demand for honey is affected by the problem of its adulteration. Moreover, despite its numerous taste and health benefits, honey may be an undesirable product for some groups of consumers, such as people with allergies or vegans. This work aimed to develop a sensitive molecular test enabling the unambiguous detection of honey adulteration and the identification of its trace amounts in food products. The test was based on the analysis of a fragment of the cytochrome c oxidase gene subunit I using real-time PCR with SYBR^®Green dye and melting curve analysis. The key parameter of the analysis was the melting temperature, which in the case of natural honey was within a narrow range of 74.34–75.38 °C (for its dilutions, 71.10–77.00 °C). The developed method demonstrated high repeatability and sensitivity, enabling the detection of honey presence even at a level of 0.1%. To products labelled as vegan, Tm analysis effectively distinguished samples containing trace amounts of honey from those that were truly vegan. The procedure used is simple, highly repeatable, and effective even in the case of processed products. The developed method can be successfully used to control the quality and authenticity of honey, meeting the requirements of V-Label certification. Full article

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis and is primarily transmitted via contaminated water and food. Groundwater may also serve as a potential vector for HEV transmission. This study aimed to optimize real-time polymerase chain reaction (rtPCR) for the detection of HEV, employing both TaqMan probe- and SYBR Green-based methods. A total of 12 primer sets for the TaqMan probe-based method and 41 primer sets for the SYBR Green-based method were evaluated in order to identify the optimal combinations. Primer sets #4 (TaqMan probe-based) and #21 (SYBR Green-based) demonstrated the highest sensitivity and specificity, successfully detecting HEV in artificially spiked samples at 1 fg/μL. The TaqMan probe-based method facilitated rapid detection with minimized non-specific amplification, whereas the SYBR Green-based method allowed for broader primer exploration by leveraging melting curve analysis. Despite the absence of HEV detection in 123 groundwater samples, this study underscores the value of real-time PCR for environmental monitoring and paves the way for enhanced diagnostic tools for public health applications. Full article

Background/Objectives: Autism spectrum disorders (ASDs) are neurodevelopmental conditions with early onset of clinical manifestations. ASD etiology is highly heterogeneous, with genetic factors being strong determinants of the behavioral problems and neurodevelopmental deficits. Fragile X syndrome (FXS) (OMIM #300624), caused by the transcriptional silencing of the FMR1 gene, represents the most common monogenic cause of autism. Our study included 226 boys with a diagnosis of ASD, for a systematic screening of genetic and epigenetic defects in the FMR1 gene promoter in a Romanian pediatric cohort. Methods: The methods, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and triplet-primed PCR (TP-PCR)/melt curve analysis (MCA), were chosen for their ability to detect the methylation anomalies (the former) as well as repeat expansions in the FMR1 promoter (the latter). Results: Both methods used in our screening generated concordant results, detecting FMR1 full mutation in 4 out of 226 patients (~1.8%). This yield is similar to data obtained in larger studies. Three out of four boys presented the typical clinical features, in correlation with genetic findings. Conclusions: The combined use of MS-MLPA and TP-PCR/MCA-based assay was, in our experience, useful to fully describe the genetic defects responsible for FXS. A significant variability of clinical presentations was observed in our small group of children with FXS, from mild to severe intellectual disability and from atypical to characteristic dysmorphic features, as well as various behavioral problems. Full article

Bovine trypanosomiasis, caused by Trypanosoma vivax, affects livestock productivity and is increasingly being reported in South America. This study aimed to detect and characterize Trypanosoma spp. infections, with a focus on T. vivax, in cattle from two distinct agroecological regions of central Argentina: a dairy-producing plain, located in the Espinal ecoregion, and a riparian zone, dedicated to beef production, located in the Delta and Islands of Paraná ecoregion. A total of 220 blood samples were collected from nine cattle farms and analyzed using real-time PCR, melting curve analysis, and the sequencing of 18S rRNA gene fragments. Trypanosoma vivax was detected at low prevalence (2.73%), exclusively in dairy cattle. In contrast, the prevalence of Trypanosoma theileri was much higher (10.91%), and it was found mainly in beef cattle from the riparian region. Phylogenetic analyses confirmed the species identity in all sequenced samples. No trypanosomes were observed by microscopy, and none of the animals showed clinical signs. The results indicate a differential distribution of T. vivax and T. theileri between regions and production systems. Although the study initially focused on T. vivax, the detection of T. theileri highlights the need to consider multiple Trypanosoma species in epidemiological surveys. This study contributes new data on the occurrence of bovine trypanosomes in central Argentina under extensive and semi-intensive management systems. Full article

Background: In recent years, numerous recurrently mutated genes have been identified in acute myeloid leukemia (AML), some of which, such as FLT3 and IDH1/2, serve as therapeutic targets, offering new treatment options. Rapid mutational analysis is crucial for timely and optimal therapy selection. This study aims to develop and validate a rapid, cost-effective, and sensitive screening method for detecting IDH1, IDH2, and FLT3-TKD2 mutations using polymerase chain reaction (PCR) and high-resolution melting curve analysis (HRM). Methods: A PCR-HRM assay was developed to simultaneously detect mutations in IDH1, IDH2, and FLT3-TKD2. The method was applied to a cohort of 1363 AML patients, and its performance, including turnaround time, was evaluated through comparison with next-generation sequencing (NGS) results. Results: The PCR-HRM method demonstrated a positive percent agreement of 98%, 98%, and 92% for IDH1, IDH2, and FLT3-TKD2, respectively, and a negative percent agreement of 100% for all three genes compared to NGS. No false positives were observed, and false negatives were detected in less than 1% of cases, mostly in FLT3-TKD2, all occurring below the established limit of detection. The turnaround time and cost of PCR-HRM were significantly lower than those of NGS. Conclusions: This method offers a highly sensitive, specific, and time-efficient approach for the simultaneous detection of IDH1, IDH2, and FLT3-TKD2 mutations in AML patients. Its rapid turnaround time and cost-effectiveness make it a valuable tool for routine clinical screening, facilitating timely and targeted treatment decisions. Full article